The present invention relates to a method of analysing a sample of free cells, in particular blood cells.
Automated block analysers which count and size blood cells, represent a huge advance in the field of chemical medicine, but retain some drawbacks. They are inherently incapable of differentiating like sized particles. While the automated count may be correct in terms of the total number of particles, traditional methods do not count small red cells, parasites or bits of cells as platelets. Anyone in need of good quality platelet counts is well advised to travel to Africa or Asia which, having few automated blood analysers, provide better quality complete blood counts because manual methods of counting avoid several errors which are common concomitants of automated sizing and counting instruments. Errors in counting platelets have serious consequences for the patient as they may result in unnecessary tests, inappropriate treatment, or mis-diagnosis. The known causes of spurious low platelet counts are EDTA dependent clumping, cold platelet agglutination, platelet satellitism and the presence of like sized particles which are not platelets.
All haematology texts, laboratories and manufacturers have been aware of the inaccuracies of automated platelet counts for many years and advise a manual inspection of a blood film for every patient with an abnormal platelet count, which is the practice in most haematology laboratories. Currently manufacturers are attempting to reduce errors by detecting light refraction (since platelets are more refractile) or by detecting stains applied to platelets before they reach the sensor. The practice of manual verification whenever the platelet measurements fall outside the normal range is comforting to the patient but expensive for the laboratories. However, hitherto, there was no method of alerting labs of the need for manual verification for patients with high platelet counts that erroneously fell inside the normal range. Thus automated platelet counts are of limited value when platelet counts are low, they are of uncertain value when the counts are normal, and are not entirely secure when the counts are elevated (thrombocytosis).
As will be discussed below, the applicants have discovered that there are other small particles, cell fragments, which are also not detected and properly distinguished by existing automated blood cell analyzers. The importance of cell friability or the generation of cell fragments has hitherto not been recognised.
According to one aspect of the present invention, there is provided a method of determining a measure of the number of platelets in a cell suspension containing platelets, the method comprising the steps of: counting the number of small particles in the suspension; agitating the suspension in the presence of a gas; counting the number of small particles in the suspension after agitation; and comparing the two counts to obtain a measure of the number of platelets.
The method of the invention differentiates platelets from other small particles exploiting a simple physiological property and removes spuriously low automated platelet counts. The accurate measurement of platelet number and function is of great consequence to a patient""s health. Aside from bleeding to death from thrombocytopenia (insufficient platelets), platelets are risk components of strokes, heart attacks, and inflammation, and are important elements in the growth of epithelial malignancies and metastases.
All red cells are very sensitive to osmotic stress, and most cells are sensitive to mechanical stress. It has been found that platelets are relatively insensitive to mechanical and osmotic stress except when exposed to contact with air. Platelet suspensions alter their properties upon exposure to air, or its component gases to a greater extent if they are simultaneously stressed. By carefully controlling the handling of platelets before and during testing, by eliminating or regulating a suspension""s exposure to air, more accurate platelet counts can be obtained. By intentionally exposing platelets to air while subjecting them to stress any induced change in the platelet population can be recorded. Because existing methods ignore the effect of air, they are inducing errors in their platelet counts.
The methods described in WO 97/24598, WO 97/24599 and WO 97/24601 provide a way to measure the size, shape, and number of particles while the particles are simultaneously exposed to a variety of osmotic gradients. However, further information may be derived from this test by combining it with the present invention. For example, by testing a whole blood suspension under osmotic stress, and comparing the results to the same sample after mechanical agitation in the presence of air, the components of the sample population which are altered by mechanical agitation in the presence of air can be determined. Furthermore, the types and proportion of each type can be revealed in a single procedure. Thus, red cells, white cells, micro-spherocytes, and bacteria are uninfluenced by mechanical agitation in the presence of air whereas platelets alone disappear. The advantage of this method in that in addition to explaining a way to differentiate and count platelets, it offers existing instruments a very simple method of increasing the accuracy of their counts.
In this application we also disclose a method of measuring cell friability since mechanical stress induces fragmentation in those cells. When cells are counted in cell counters, neither lysis nor fragmentation nor ghosts are induced. When cells are subjected to osmotic stress they lyse and ghost or fragment or both. The addition of mechanical agitation enhances the effect and the addition of air into the agitated suspension further intensifies the effect. Thus, in order to differentiate between cell fragments and platelets, it has been found to be preferable to compare the count of small particles before and after agitation in the presence of air against osomolality. This process of inducing the nature of these small particles can be likened to the identification of an unknown fluid by raising its temperature; if it boils at 78xc2x0 C. it is ethyl alcohol, if it boils at 100xc2x0 C. it is water and at 357xc2x0 C. it is mercury.
In order to provide quantative measures from the present invention, it is preferable to monitor at least one of the following: A) the air quantity in contact with the suspension, B) the intensity of agitation and C) the duration of agitation, and to relate this measured value to the difference between the two counts.
The small particles counted in the method of the invention include platelets, bacteria, cell fragments, and micro-spherocytes, which typically have a volume of 7-10 femtoliters.
According to another aspect of the present invention, a method of analysing a sample of free cells in vitro comprises applying a known or identifiable quantity of stress to the sample, measuring the sample before and after the application of stress to provide at least one reading from which quantitative information relating to the number of cell fragments in the sample caused by the applied stress can be determined, and relating this reading to the quantity of stress to provide an indication of cell friability.
When red cells die, they lose their contents, a process termed lysis. They are then transformed into either ghost cells or fragments depending, in part, on the cell""s membrane properties, and in part on the provocation. Hitherto, this mechanism has been little recognised or understood.
The present invention is based on the realisation that if cells, such as blood cells, and in particular red cells, are stressed in vitro the relationship between the applied stress and fragmentation to the sample is characteristic for normal samples and for many diseases. Since some cells (platelets) produce no detectable fragments and some (red cells) produce a large number this method also provides a way of distinguishing between certain cell types.
Existing automated particle analysers are of limited use in detecting fragments as they cannot identify fragmentation that was cleared in vivo by increased phagocytosis, and when fragments were not phagocytosed, it cannot easily differentiate them from platelets, apoptotic bodies, micro-spherocytes, parasites and noise. There are many patients whose cells are fragmenting yet no fragments are detected by existing methods because the body""s physiological clearing mechanism removes fragments as fast as they are produced.
The method of the present invention has been found to be remarkably good at identifying certain blood abnormalities because it detects patients whose cells fragment when stressed. Because applied stress affects red blood cell fragments and platelets differently, it is possible to distinguish between the two. This has not been possible in the prior art particle counters because platelets and red blood cells fragments are usually not measured individually. In addition, as a sample can be tested before, during and after a stress has been applied, the induced effect provides an indication of the physiological fragmentation potential and the physiological removal rate.
The invention can be used to identify the age of population of cells since old cells fragment readily whereas new ones do not fragment readily. This may be useful in blood-banking, for instance, to destroy selectively a particular population of cells using a particular range of stresses, thereby prolonging the life of the unit by culling the old cells.
The applied stress can be mechanical, chemical, thermal, sonic, light, electric, electromagnetic, or any other means which induces membrane stress including in vitro aging of the cell sample. Preferably, the cells are subjected to an osmotic gradient. They may also be agitated by a stirring bar, by shaking vigorously, or by any other type of stress. More than one stress mechanism can be used at the same time if necessary. Indeed this may be beneficial in identifying certain abnormalities.
It is, however, preferable to apply a known stress, and more preferable to apply a range of stresses, which may be decreasing or haphazard, but is preferably increasing, so as to obtain a plot of the relationship between the number of red blood cell fragments and the applied stress. This range of stresses can be applied by varying duration and/or intensity, for example, by virtue of mechanically agitating the sample increasingly vigorously, by mechanically agitating the sample at a constant rate over an increasing period of time, by diluting the sample with a solution which gradually decreases in osmolality, or by any combination of these mechanisms. The latter approach can be carried out using the apparatus disclosed in WO 97/24529, which generates an osmolality gradient.
Characteristics providing the quantative information relating to the number of cell fragments include the detection of the fragments themselves, an alteration in the frequency distributions of multiple cell populations, an increase in the total particle count, a change in the concentration of intact cells, a change in the concentration of membrane or cytosolic parts, such as haemoglobin release, or other such phenomena related to induced cell fragmentation.
The step of counting the number of small particles is preferably done using a conventional commercially available particle counter, or using the apparatus disclosed in WO 97/24600. In both cases, the blood sample is caused to flow through a sensor, typically an aperture, where its size is detected optically, acoustically, thermally, electronically or by other means. In an impedance sensor, the response of the electrical field to the passage of the cells is recorded as a series of voltage pulses, the amplitude of each pulse being a function of cell fragment size and frequency.
In the absence of applied stress, existing instruments are unable to distinguish between platelets and, for instance, red cell fragments. As each different cell type has its own biochemical individuality and its own sensitivity to fragmentation, it is possible to use its response as an identifying property, identifying the cell type, sensitivity and pathology. For instance, platelets which are-similar in size to fragments, are differentiated from fragments as they respond differently when stressed. The differentiation and identification of fragments, platelets, ghost cells and other cell parts may be facilitated by using stains and dyes which selectively dye cell lines, or bond onto specific cell parts, for instance the inside and outside of the cell membrane or only stain the platelets. When a stress has been applied to a blood sample, as for example, in WO 97/24598, WO 97/24599 and WO 97/24601, it was performed to obtain accurate volume and other cell measurements by forcing cells to a known shape. The method disclosed herein induces cells to fragment and measures the result of fragmentation. This may be enhanced by, for instance, eliminating all particles except for the fragments of interest by setting the upper and lower threshold voltages to the size range of fragments, by facilitating fragmentation by applying heparin or other suitable chemicals in the chemical preparation of the sample and/or by allowing sufficient time for fragmentation to proceed. The induction, detection and quantification of fragments enhances the measures produced by WO 97/24598, WO 97/24599 and WO 97/24601 and other measures which may be altered by induced or existing fragments.
When using an electrical particle counter, the thresholds should be set low enough to detect cells down to a volume of almost 0 femtoliters. A threshold voltage of 0.08 mV has been found to work well with the existing apparatus. Other populations of particles may be eliminated electronically, digitally, mechanically, or by other means.
The commercially available particle counters and the device disclosed in WO 97/24600 can be used to provide adequate readings for the present invention. However, more accurate readings can be obtained if the size of the aperture in either device is reduced so that the ratio of the cross section of the aperture to the mean cross section of the red blood cell fragments or platelets is substantially 4:1.
In conventional blood cell analysis techniques, it is usual to treat the sample with an anticoagulant, such as EDTA, prior to analysis. However, it tends to inhibit fragmentation. It has therefore been found preferable not to treat the sample with an anticoagulant or to use an anticoagulant which does not inhibit or promote fragmentation, such as heparin.